Abstract

The
aim of the study was to determine the anthelmintic activity of Cassiaabbreviata, Schotia brachypetala, Senna italica,
Pappea capensis and Peltophorum africanum against egg and third
larval stages of H. contortus. The bark of C. abbreviata,
P. capensis and P. africanum, the root bark of S. italica
and the leaves of S. brachypetala were harvested, dried at room
temperature in the laboratory and thereafter extracted in hot and cold
distilled water. Concentrations of 2.5, 5.0 and 7.5 mg/mL of these extracts
were tested for inhibitory activity against H. contortus egg hatching
and larval development.

Hot and cold water extracts of P.
africanum inhibited the highest percentage of eggs (25 and 22%,
respectively) from hatching at the lowest concentration of 2.5 mg/mL. These
extracts did also inhibit 86 and 80% of larval development, respectively.
The highest rates of larval mortality were recorded for P.
africanum and S. italica hot water extracts(68%
and 65% within two hours at the lowest concentration and 63% and
64 % for cold water extracts. The percentage of larval mortality increased
as the concentrations increased. However, the experimental results were not
solely dependent on extract concentrationbecause all the third stage
H. contortus larvae died within 72 hours irrespective of the level of
concentration. This work therefore supports the use of the five experimental
plant species used in the present study for the control of gastrointestinal
nematodes by the Bapedi people of the Blouberg Municipality in Limpopo
Province of South Africa and recommend that further studies like toxicity
testing and in vivo trials be made to validate the use of these
plants as an alternative to anthelmintic drugs.

Introduction

Internal parasite infestations constitute a major constraint to sheep
production in South Africa (Perry & Randolph 1999), with Haemonchus contortus
considered the most important of all the gastrointestinal nematodes that
restrain the survival and productivity of sheep and goats owned by the rural
poor in the developing world (Perry et al 2002). The control programmes for
H. contortus usually result in the control of other internal
worms (Kandu-Lelo 2010).

In
Africa, commercial anthelmintics are expensive and sometimes unavailable
leading to the use of poor quality or altered products (Luseba & Tshisikhawe
2012). The misuse of these synthetic medicines has led to the
development of anthelmintic resistance (Lans & Brown 1998). According to
Kaplan (2004),H. contortus features prominently amongst the reports of
anthelmintic resistance that has emerged in all countries of the world that
produce small ruminants. In Africa, anthelmintic resistance has been
reported in both the commercial and resource poor farming sectors (Tsotetsi
et al 2013). This justifies an urgent need to find alternatives to synthetic
drugs (Shen et al 2010) such as ethnoveterinary remedies. South Africa’s
indigenous people have the knowledge of plant species with anthelmintic
activity. Furthermore, because of limited availability of drugs, high cost,
development of resistance, chemical residue in milk and meat, the majority
of world population depends on traditional remedies (Jeyathilakan et al
2011).

Farmers claim that medicinal plants are more effective than pharmaceuticals
for chronic pathologies (Luseba et al 2007). Luseba and Van der Merwe (2006)
reported the use of ethnoveterinary medicines by Setswana-speaking people in
the Madikwe area of the North West province and the Tsonga speaking people
of the Limpopo Province, respectively. However, ethnoveterinary medicine has
proven to be ethnical and locality-specific (Luseba & Van der Merwe 2006).
Thus, research findings in other areas might not be applicable to the
present study area. Therefore, the aim of this study was to investigate the
anthelmintic activity of plant species used to treat parasite infections of
livestock by Pedi-speaking communities in the Blouberg Municipality of the
Limpopo Province, South Africa for the preservation of local knowledge by
way of documenting it.

Materials and Methods

Study
area

Interviews and plant collections were conducted in the Blouberg Municipality
(Figures 1 (a) and 1(b) which falls under the Capricorn District
Municipality and comprises the arid sweet bushveld (Acocks 1988) in the
Limpopo Province. Rainfall is 400 mm per annum and the rainy season usually
extends from November to February but rainfall distribution is irregular and
unpredictable. Average minimum and maximum temperatures are 12░ C and 25░ C,
respectively (Mara research station, South Africa). A permit (Permit no. RB
102/13) for collection of plant materials was obtained from the Limpopo
Department of Environmental affairs in Polokwane before plant specimens of
each species were collected, labelled and pressed according to the methods
of Fish (1999).

Questionnaire survey

Purposeful sampling of homogenous groups where participants are most likely
to give good insight of the phenomenon of interest was conducted (Patton
1990).
A total of 60 livestock farmers with profound knowledge on plant species
used for the treatment of internal parasites were interviewed.
Many rural small stock farmers in Blouberg Municipality fall outside the
periphery of formal livestock markets. However, statistics show that small
stock rearing remains an integral part of their everyday life and it is an
important source of income (Grwambi et al 2006). Semi-structured interviews
were conducted using an open-ended questionnaire between May and August 2013
which aimed at collecting data relating to tree and shrub species that are
used against helminthosis by livestock farmers in the Blouberg Municipality
of the Limpopo Province. Areas of discussion were guided by descriptions
such as farmer’s age, gender, socio-economic profile, animal husbandry and
local knowledge in animal healthcare (Luseba & Van Der Merwe 2006).
Comprehensive information of plants used by the local people such as local
name, indications, method of preparation, administration and dosage to
formally record the ethno-botany of the area was recorded.

Plant
material and preparation of extracts

Plants
cited more than two times as having anthelmintic properties by interviewed
farmers were selected for the current study (Kansonia and Ansay 1997). They
were collected and thereafter authenticated by a botanist (Dr. B Egan) at
the University of Limpopo. In order to have an appropriate quantity of
samples for laboratory analyses, approximately 1-2 kg of fresh plant
materials were collected for each plant species and from two or more shrubs
or trees around Blouberg Municipality of the Limpopo province (33░47′50″S
18░27′43″E). The plant materials (leaves, bark and root-bark) were collected
from October to December 2013, dried at room temperature in the laboratory
and weighed frequently on a daily basis to assess the moisture content until
constant weight was obtained after subsequent weighing. They were then
ground to fine powder using a Macsalab mill (Model 200Lab, Eriez«, Bramley,
RSA). Five grams of each powdered material was extracted in hot and cold
water at 10 mL/g respectively overnight. The extracts were filtered using
Whatman« No.1 filter paper (Whatman, United Kingdom) and filtrates were
frozen at -80oC before drying using a Lyoquest 50 freeze dryer (Labotec,
South Africa). The extracts were reconstituted in distilled water for their
respective stock solutions. Then, the stock solutions were diluted to the
required concentrations of 2.5, 5.0, and 7.5 mg/mL for the bioassay
analyses.

Parasite sample collection

According to Kandu-Lelo (2010) the control programmes for H.
contortus usually result in the control of other internal worms, hence
results derived from experiments using H. contortus as a model can be
applied to other nematode species. Faecal samples were collected directly
from recta of adult ewes experimentally infected with H. contortus
field strain. The sheep belonged to Dr. P.C Van Schalkwyk of Biozetica Agri-Source
(Pty) LTD and were kept on plot A 64 Buffelshoek, Mooinooi near Rustenburg
in the North West Province of South Africa (25░79′08″ S 27░55′15″ E).
Collected samples were immediately transported to the Helminthology
laboratory of the Agricultural Research Council, Onderstepoort Veterinary
Institute in a cooler box. Faecal samples were analysed using the McMaster
technique (Soulsby 1982) to confirm the presence of nematode eggs and faecal
cultures prepared (Reinecke 1973) to identify/confirm the nematode genera
using Van Wyk et al (2004).

Biological assays

Four
assays were conducted. In order to determine the anthelmintic activity
of the prepared plant extracts, Haemonchus eggs were recovered from
faeces through an egg recovery assay (Maphosa et al2010) with some
modifications.

The
egg hatch assay was conducted as published by McGaw et al (2007) with some
minor modifications. The larval development assay was conducted as described
by Bizimenyera et al (2006).

Larval
mortality assay was conducted according to the method described by McGaw et
al(2007) with some modifications. All live and motile L3s
in each well were counted and mortality was expressed as a percentage. All
tests were replicated three times.

Results

Farmers’ profile

The
highest frequencies of trees and shrubs were cited by males compared to
females as shown in Table 1. Older people aged over 40 years, of which 20
(33.3%) were females and 40 (66.6%) males, reported to know more plant
species used to treat internal parasites in livestock than younger people.
All in all, the number of females aged under 40 years were 5 which is 8% of
the total number of participants and 25% of the female participants whereas
on the other hand the number of male participants under the age of 40 were
15 which is equal to 25% of the total number of people who took part in the
study and 37.5% of the male participants.

Table 1.
Frequencies of trees and shrubs cited by both male and females of different age groupsin Blouberg Municipality

Plant

Females

Males

Freq: 21-40 years

(%)

Freq: Above 40 years

(%)

Total Freq.

(%)

Freq: 21-40 years

(%)

Freq: Above 40 years

(%)

Total Freq.

(%)

Peltophorum africanum

4

44.44

7

29.1

11

33.33

6

40

25

27.77

31

29.5

Senna italica

2

22.22

4

16.66

6

18.18

1

6.67

19

21.1

20

19.05

Cassia abbreviata

1

11.11

3

12.5

4

12.12

1

6.67

12

13.33

13

12.38

Schotia brachypetala

1

11.11

4

16.66

5

15.15

1

6.67

9

10

10

9.52

Pappea capensis

1

11.11

2

8.33

3

9.09

2

13.33

8

8.88

10

9.52

Ochna pulchra

0

0

1

4.16

1

3.03

1

6.67

4

4.44

5

4.76

Capparis sepiaria

0

0

1

4.16

1

3.03

1

6.67

3

3.33

4

3.81

Mormodica balsamina

0

0

0

0

0

0

1

6.67

2

2.22

3

2.86

Jethropha zeyheri

0

0

0

0

0

0

0

0

3

3.33

3

2.86

Dicerocarym senecioides

0

0

1

4.16

1

3.03

0

0

2

2.22

2

1.9

Gymnosporia senegalensis

0

0

0

0

0

0

0

0

2

2.22

2

1.9

Punicum granatum

0

0

1

4.16

1

3.03

1

6.67

1

1.11

2

1.9

Where Freq. = Frequency; % = percentage of frequency

Table 2 shows the thirteen plant species belonging to seven families that were
identified of which the plant family containing more species was the
Fabaceae. The five plants that were used for biological assays were selected
because according to Kansonia & Ansay 1997, there is consistency when one
plant is cited for the same use by more than two respondents.

Table 2.
Plants and plant parts administered for the treatment of internal parasites in animals in the Blouberg Municipality

Biological assays

Effect of cold and hot water plant extracts on H. contortus egg hatching

The
highest inhibition of 22 and 25%, at the lowest concentration of 2.5 mg/mL,
were recorded for P. africanum cold and hot water extracts,
respectively whereas P. capensis showed the lowest inhibition
percentages of all the tested plants. Generally, the egg hatch assay
indicated that hot water extracts significantly inhibited (p = 0.05) the
hatching of a higher number of eggs compared to the cold water extracts
except for P. capensis 2.5 mg/mL and 5.0 mg/mL where there was no
significant difference (p=0.05) between cold and hot water plant extracts
(Table 3).

Table 3.
Mean inhibition percentages for the egg hatch assay for Haemonchus contortus, using different concentrations (conc.) of crude
cold and hot water extracts of five plants

Plant

Cold water

Hot water

Controls

Conc. (Mg/ml)

2.5

5.0

7.5

2.5

5.0

7.5

+

-

C. abbreviata

13.00±0.00s

19.00±0.00op

37.00±1.00g

15.33±1.52qr

21.00±0.00no

40.00±1.00f

100

0

S.brachypetala

14.66±2.08rs

23.33±1.52lm

39.00±2.00fg

17.00±2.00pq

25.66±1.52k

42.66±1.52e

100

0

S. italica

17.66±1.52p

28.66±0.57j

47.66±0.57d

20.66±0.57no

32.00±1.00i

53.00±0.00c

100

0

P. capensis

5.33±1.52u

11.00±2.00t

13.66±0.57rs

7.33±1.52u

13.00±1.00st

17.66±1.52p

100

0

P. africanum

22.00±2.00mn

31.00±2.00i

55.33±0.57b

25.00±0.00kl

34.33±1.52h

58.00±1.00a

100

0

Means with the same letter (a-u) are not significantly different (p =0.05). + = Positive; - = Negative

Effect of cold and hot water plant extracts on Haemonchus contortus larval development and viability

The
results for larval development and viability are reported in Table 4.
Similar to the egg hatch assay, the larval development assay indicated a
linear dose related inhibitory response.
The highest inhibition of larval development at the lowest concentration was
observed for P. africanum with 80.00 and 86.33% for cold and hot
water plant extracts respectively whereas the lowest inhibition percentage
at the lowest concentration was observed for P. capensis with 66.00
and 69.00% for cold and hot water plant extracts respectively. There was a
significant difference between P. africanum cold and hot water
extracts while the same was observed for S.italica (p =
0.05). The
positive control thiabendazole induced a 100% larval development inhibition
whereas the negative control inhibited 0% of pre-infective L1 and
L2 larvae from developing to the parasitic infective L3
larvae stage. The
plant extracts proved to be much more effective against the infective stage
larvae than with respect to the pre-infective stage larvae.

Table 4.
Mean inhibition percentages for the larval development assay for Haemonchus contortus, using different concentrations (conc.)
of crude cold and hot water extracts of five plants

Plant

Cold water

Hot water

Controls

Conc. (Mg/ml)

2.5

5.0

7.5

2.5

5.0

7.5

+

-

C. abbreviata

69.00±0.00k

75.00±2.00ij

87.66±1.52ef

74.33±0.57ij

83.00±2.00g

91.66±2.51c

100

0

S.brachypetala

70.33±1.52k

80.66±1.52h

90.33±1.52cd

76.00±1.00ij

86.00±0.00f

95.66±0.57b

100

0

S. italica

76.33±0.57i

86.66±0.57f

100.00±0.00a

80.00±0.00h

89.00±2.00ed

100.00±0.00a

100

0

P. capensis

66.00±2.00l

70.33±1.52k

76.00±0.00ij

69.00±2.00k

74.00±0.00j

80.00±2.00h

100

0

P. africanum

80.00±1.00h

89.00±0.00de

100.00±0.00a

86.33±1.52f

90.33±3.05cd

100.00±0.00a

100

0

Means with the same letter (a-l) are not significantly different (p =0.05). + = Positive; - = Negative

Effect
of cold and hot water plant extracts on larval mortality

The results for the larval mortality assay with hot and cold water extracts.
The larval mortality rate was dose dependent in both hot water and cold
water extracts. The highest rates of mortality were recorded for P.
africanum and S. italica cold water extracts,with 63%
mortality and 64% mortality rates within two hours at the lowest
concentration, and 68% and 65% for hot water extracts respectively. The
positive control Thiabandazole« killed all the L3 larvae within 2
hours of exposure even at the lowest concentration of 2.5 mg/mL, while as
expected there was no larval mortality in the negative control, which was
distilled water. All
the hot water plant extracts recorded the highest inhibition percentages
compared to all the cold water plant extracts at all concentrations.

About
72 hours were needed to result in a total larval mortality irrespective of
the concentration. The percentage of mortality increased as the
concentrations increased;however, the experimental results were not
concentration dependentbecause all the third stage H. contortus
larvae died within 72 hours at2.5, 5.0 and 7.5 mg/mL, respectively
(Table 5). Even at the lowest concentration of 2.5 mg/mL in both cold and
hot water there was no extract from all five of the tested plants that
induced larval mortality of less than 50 percent. No significant difference
was found to exist in all the plant extracts, and in all the three different
concentrations, at the observation time of 72 hours (p = 0.05).

Discussion

The
frequencies of plants mentioned during the survey coincide with the anthelmintic
activities of the plants against H. contortus. For instance P.
africanum was mentioned more often than any other plant for the treatment of
worm infestations in animals, while the biological assays revealed that P.
africanum was the most potent plant; on the other hand, P. capensis
which was the least popular plant was not as effective as P. africanum
against the H. contortus at three different stages of development. There
was also no significant difference (p=0.05) on the low inhibition percentage of
both cold and hot water P. capensis plant extract for egg hatch assay.

The
results from the egg hatch assay signify that, although egg hatch inhibition was
observed, not all eggs were inhibited from doing so. Egg hatch inhibition
percentage for P. africanum cold water extract which proved to be the
most potent plant extract which recorded 22 % at the lowest concentration and 55
% at the highest concentration whereas hot water plant extract of the same plant
inhibited 25% at the lowest concentration and 58% at the highest concentration
Although there was some inhibition, most eggs managed to hatch and according to
Molefe el al (2012) this might be because the egg is, at this stage,
disseminated into the environment and protected with a thick wall, causing it to
be resistant to various environmental conditions.

This
results of the present study suggests that P. africanum and S. italica
are still effective
in preventing the pre-infective larvae from developing to the infective L3
larvae stage
even at low concentrations. As expected, there was a significant difference
between 2.5, 5.0 and 7.5 mg/mL concentrations in both hot and cold water plant
extracts for egg hatch and larval development assays.

The most
outstanding feature of the larval mortality assay results was that there was no
significant difference (p=0.05) between the cold and hot water plant extracts in
all the concentrations at 72 hours which could mean that the longer the worms
remain in contact with the plant extracts, the lower their chances of survival.
Although the results from the present study supports the theory that P.
africanum has an ovicidal effect on H. contortus eggs, no significant
difference was found between P. africanum cold water extract and S.
italica hot water extract; furthermore, no significant difference was
established between S. brachypetala cold water extract and C.
abbreviata hot water extract irrespective of the concentration (p = 0.05).

There was
no significant difference between P. africanum 2.5 mg/mL cold water
extract and S. italica at the concentration of 2.5 mg/mL hot water
extract for the larval development assay as well. Similarly, there was no
significant difference for egg hatch inhibition (p = 0.05) between P.
africanum 2.5 mg/mL hot water extract and S. brachypetala 5.0 mg/mL
hot water extract, nor between S.italica 2.5mg/mL hot water extract,
P. africanum 2.5 mg/mL cold water extract and Cassia abbreviata 5.0
mg/mL hot water extract. In other words, P. africanum and S. italica
were effective even at low concentrations.

Even though P. africanum proved to be the most potent of the five plant
extracts, all these (both hot and cold water extracts) managed to inhibit
further development of the free living pre-infective L1 and L2
larvae into the infective L3 larvae,and the result was
100% mortality within 72 hours. It was also evident that hot water extracts were
responsible for higher rates of larval mortality in all the treatments. The
efficacy of any plant extract, at the lowest concentrations, against the
gastrointestinal nematodes proves the anthelmintic activity of that plant (Maphosa
et al 2010). It is therefore concluded that
C.abbreviata,
S. brachypetala, S. Italica, P. cappensis P. africanum
exhibit such an activity. It is also important to note that the survey revealed
water as a common solvent used in the preparation of concoctions to be given to
the animals for the treatment of internal parasites, which concurs with what
Bizeminyera et al (2006) reported. The high polarity of the bio-active compounds
in the plants also means that these compounds may be extractable by the polar
solvents available to rural users.
Molefe (2013) also reported the high effectiveness of water extracts compared to
acetone extracts for the egg hatching and larval mortality assay.

On one
extreme, P. africanum proved to be the most potent plant while on the
other, P. capensis was the least potent, as determined by the highest egg
hatch and larval development inhibition percentages and the highest larval
mortality percentage at the lowest extract concentration of 2.5 mg/mL with
minimal contact time of 2 hours between the larvae and the plant extract. On the
intermediate level were S. italica, S. brachypetala and C.
abbreviata, respectively.

It is clear that P. africanum bark and S. italica root-bark
recorded high anthelmintic activity and are
therefore good candidates for treatment of gastrointestinal infections. However,
the mechanisms of their effectiveness still remain to be tested in vivo.
Furthermore, safety and toxicity studies must be conducted in vivo to
determine the minimum non-lethal concentrations needed for the treatment of
nematode infections.

Overall,
the study revealed that the hot water extracts of all tested plants were more
effective than cold water extracts with respect to the egg hatch inhibition,
larval development and larval mortality assays. Therefore,
hot water plant extracts of P. africanum bark and S.italica
root-bark could be considered as a replacement for synthetic drugs. However,
S. brachypetala, C. abbreviata and P. capensis exhibit
moderate anthelmintic activity; as a result, they cannot be used as a sole
replacement for synthetic drugs but rather as an integrated approach to achieve
sustainable parasite control in ruminant production systems (Githiori et al
2006).

Adamu et
al(2013) stated in their report on work done on the efficacy and
toxicity of thirteen plant leaf acetone extracts used in ethnoveterinary
medicine in South Africa, as regards egg hatching and larval development of H.
contortus, that to their surprise the aqueous extract of Markhamia
obtusifolia displayed double the activity of the acetone extract. According
to these authors these results indicate that the anthelminthic activity of
aqueous extracts of plants which had already been investigated using organic
solvents should be determined.
The results of the current work are crucial in advancing towards finding long
term solutions in alternative treatment for since control programmes for
Haemonchus contortus usually result in the control of internal worms (Kandu-Lelo
2010).

Conclusion

Hot
and cold water extracts of P.
africanum inhibited the highest percentage of eggs from hatching at the
lowest concentration. These extracts also exhibited high inhibition
percentages of larval development. The highest rates of larval mortality
were recorded for both P. africanum and S. italica
hot and cold water extractsat the lowest concentration.
This work therefore supports the use of these plant species in the control
of internal parasites by the Bapedi people of the Blouberg Municipality in
Limpopo Province. This is particularly the case because any validation of
the use of plants in traditional medicine which is based on the results
obtained using organic solvents might be considered irrelevant since,
traditionally, water is used instead as a solvent for most of the
preparations.

Luseba
D, Elgorashi E E, Ntloedibet D T and Van Staden J 2007
Anti-bacterial, anti-inflammatory and mutagenic effects of some medicinal plants
used in South Africa for the treatment of wounds and retained placenta in
livestock. South African Journal of Botany. Volume73:378-383.